The present invention relates to the use of an internal positive control containing an inverse sequence to detect inhibition and to provide an internal quantitation standard in a nucleic acid assay.
Modern nucleic acid assay techniques allow researchers and clinicians to detect molecules of interest that are present in extremely low concentration. These assays use probes to specifically amplify by several orders of magnitude and detect the amount of the molecule of interest. However, when used diagnostically, falsely negative results arising from inhibition of the assay reaction dramatically reduce the predictive value of the assay. Thus there is a strong need for a method to control for inhibition of the assay reaction.
The Polymerase Chain Reaction (PCR) is an example of such an amplification technique for the detection of target molecules. With PCR it is possible to test blood samples for minute quantities of nucleic acid from pathogens, such as the human immunodeficiency virus (HIV). The technique can also be used to detect a variety of different infectious agents in a number of different clinical settings such as testing blood or donor organs for infection. Negative results may be unreliable given the susceptibility of these techniques to non-specific inhibition by a variety of compounds. Thus, there is a requirement for methods to differentiate true negative results from false negative results secondary to inhibition of the assay.
For the foregoing reasons, there is a need for an accurate reproducible positive control to detect inhibition in the PCR reaction. The method of detecting inhibition is further applicable to other signal amplification assays.
The present invention relates to compositions and methods that provide a positive control to identify inhibition during a signal amplification reaction. The methods and compositions of the present invention are designed to run in the same tube or assay environment as the experimental or target sample and contain a copy of the target sequence in an inverted form.
One embodiment of the present invention, provides for an internal control cassette for use in a polynucleotide detection assay in which a target sequence is detected. The target sequence has primer binding sites flanking an internal target sequence. The cassette comprises primer binding sites flanking an internal control sequence, wherein the internal control sequence comprises said internal target sequence in a reversed orientation.
In one aspect of this embodiment, the internal control cassette further comprises one or more primer binding sites adjacent to the internal control sequence. In another aspect, the internal control cassette further comprises the nucleic acid sequence of SEQ. ID. NO. 5. The internal control cassette may be a component of a plasmid.
Another embodiment of the present invention contemplates a method for detecting signal amplification inhibition in an assay comprising the steps of: co-amplifying a target sequence and an internal control cassette, wherein the internal control cassette comprises the target sequence in a reverse orientation. Assays contemplated for use with the present invention are selected from the group consisting of PCR, real-time PCR, branched DNA (bDNA)-based signal amplification assays, nucleic acid sequence based amplification assays (NASBA), and transcription mediated amplification (TMA).
The target sequences usable in the present invention include any nucleic acid sequence that may be assayed with techniques known in the art. In one aspect of this embodiment, the target sequence comprises DNA or RNA. In another aspect, the target sequence is a nucleic acid sequence from a virus selected from the group consisting of HSV, HIV, HCV, CMV, and HPV. In another aspect, the target sequence comprises the nucleic acid sequence of SEQ. ID. NO. 1.
Similarly, the internal control cassette sequences include any sequence that may be a target sequence. For example, an internal control cassette comprises the nucleic acid sequence of SEQ. ID. NO. 2.
The methods of the present invention further comprise the step of assaying products generated by the co-amplification described above. The present invention further contemplates an additional step of assaying products by a primer binding assay comprising the step of determining the extent of product binding to a capture probe specific for the internal control cassette product. In one aspect of this embodiment, the capture probe consists of the nucleic acid of SEQ ID NO. 6.
Another embodiment of the present invention contemplates a method for detecting signal amplification inhibition in an assay comprising the steps of contacting one or more hybridization probes with both a target sequence and an internal control cassette in the same medium, wherein the internal control cassette comprises the target sequence in a reverse orientation. For example, the assay of this embodiment is a molecular beacon assay. In another aspect, the internal control cassette comprises the nucleic acid sequence of SEQ. ID. NO. 2. In still another aspect, the hybridization probe comprises the nucleic acid sequence of SEQ. ID. NO. 8.